Synthesis and structural characterization of glucooligosaccharides and dextran from Weissella confusa dextransucrases

Many lactic acid bacteria are able to synthesize dextrans from sucrose by dextransucrases. Weissella confusa strains have attracted increasing attention due to their production of texture-modifying dextrans during food fermentation. Potential prebiotic oligosaccharides have also been produced by dextransucrases in the presence of sucrose and acceptor sugars. However, only few W. confusa dextransucrases have been previously studied, and the reactions of Weissella dextransucrases to synthesize oligosaccharides have not been investigated. In this thesis, two W. confusa dextransucrases from efficient dextran-producers were studied, and their products were structurally characterized because this information is essential for a better usability of these enzymes and corresponding bacteria in food and health applications. The biochemical and kinetic properties of one of the W. confusa dextransucrases were studied. Two activity assays were compared to determine the kinetic parameters. A sucrose radioisotope assay gave a KM of 14.7 mM and a Vmax of 8.2 µmol/(mg∙min), whereas a Nelson-Somogyi assay gave values of 13.0 mM and 19.9 µmol/(mg∙min), respectively. The dextrans from the two W. confusa dextransucrases were found by, e.g., NMR analysis to consist of mainly α-(1→6) linkages and 3% α-(1→3) branches, of which some were elongated. A high-performance size-exclusion chromatography analysis of the dextrans revealed high molar masses of 107‒108 g/mol. Weissella dextransucrases were also studied with acceptor sugars for the synthesis of glucooligosaccharides. The most efficient acceptor was maltose, followed by isomaltose, maltotriose, and nigerose, which formed series of glucooligosaccharides by the further elongation of intermediate acceptor products. The products derived from maltose formed two homologous series, with one series being predominant and the other being minor. The major maltose product series were linear isomaltooligosaccharides (IMOs) with reducing-end maltose units, as identified by multistage mass spectrometry (MSn) analysis. The minor maltose series were revealed by an NMR analysis of the isolated minor trisaccharide product to bear a novel branched structure. These products contained an α-(1→2)-linked glucosyl residue on the reducing residue of the linear IMOs. These structures have not been previously obtained by a dextransucrase. They probably formed by the attachment of a single-unit branch to linear IMOs. For the acceptor analogs lactose and cellobiose, their main acceptor products were identified by NMR analysis to be branched trisaccharides, with a glucosyl residue α-(1→2) linked to the acceptor s reducing end. Surprisingly, a side product, isomelezitose (6Fru-α-Glcp-sucrose), was produced when using lactose as an acceptor. The synthesis of this nonreducing trisaccharide by a dextransucrase was reported here for the first time. Linear IMOs with a degree of polymerization ≥3.3 serve as prebiotics for their resistance to digestion. However, industrial IMO production often leads to a high portion of unwanted digestible sugars. The dextransucrase reaction in the presence of the efficient acceptor maltose was demonstrated here as a promising alternative synthesis process to control IMO size distribution by varying the sucrose/maltose ratio. The effects of substrate concentrations (0.15 1 M) and dextransucrase dosage (1 10 U/g sucrose) on the IMO yield and profile were modeled. High sucrose (1 M) and medium maltose (0.5 M) concentrations were found to be optimal for the synthesis of long-chain IMOs, with 366 g/L of total IMOs attained.